1. Field of the Invention
The present invention relates generally to the purification of peroxidase enzymes, and more particularly, to a method for removing impurities from an aqueous solution containing horseradish peroxidase by using extraction techniques.
2. Description of the Prior Art
Peroxidase enzymes are fairly ubiquitous, occurring in higher plants, yeasts, molds, bacteria and mammals. One of the most common peroxidase enzymes is horseradish peroxidase. Horseradish peroxidase is produced from aqueous extracts of diced or ground-up whole horseradish roots, horseradish skin extracts or washings, or washings of whole horseradish.
Horseradish peroxidase has many uses. One such use is as an agent employed in immunossays. Another use of horseradish peroxidase is in the formulation of biocides. In an associated use, the horseradish peroxidase enzyme is particularly suited for its ability to catalyze the oxidation of phenolic materials (see, for example, U.S. Pat. Nos. 4,370,199; 4,478,683; and 4,647,952). As is known in the art, the oxidation of phenol typically occurs by utilizing a peroxide-peroxidase enzyme system, and more particularly, a hydrogen peroxide/horseradish peroxidase enzyme system.
Although horseradish peroxidase enzymes have been successfully used for the above applications, they are limited in their efficacy as a result of contamination. Microbial and lipophilic contamination appears to be the major contributing factor for the instability of peroxidase enzymes. Further, protease enzymes, a class of enzymes which hydrolyze proteins to amino acids and polypeptides, are water soluble products which degrade proteins and can degrade peroxidase. This too reduces the efficacy of utilizing horseradish peroxidase.
Alternatives have been proposed for purifying peroxidase enzymes. For example, U.S. Pat. No. 3,947,324 assigned to G. D. Searle discloses a method for isolating peroxidase enzyme from plant tissue containing peroxidase. The method includes treating aqueous extracts of plant tissue having a pH adjusted to 6-9 with an amount of zinc ion to form a zinc ion-protein contaminant precipitate and a supernate, removing the precipitate, and separating the peroxidase enzyme from the supernate.. The reference further discloses that the separating step comprises absorbing the concentrated supernate on carboxymethyl cellulose, eluting peroxidase from the carboxymethyl cellulose with an aqueous buffer, precipitating the peroxidase from the aqueous buffer with an organic solvent , and filtering and drying the precipitate.
U.S. Pat. No. 4,657,864 assigned to Westvaco discloses a method for stabilizing peroxidase solutions. The method described in the reference comprises selecting a peroxidase solution of low protease content or treating the peroxidase solution to reduce its protease content, and then filtering the solution through a microporous membrane to sterilize and remove from the solution any microbial contamination that might secrete additional protease enzyme. According to the reference, the filtration membrane has a pore size ranging from about 0.20 to about 0.45 microns.
U.S. Pat. No. 4,043,870 assigned to Upjohn discloses a process for purifying aqueous solutions which contain cholestrol oxidase. In particular, the reference discloses a process for removing non-ionic surfactants from a solution containing cholestrol oxidase by extraction with a water-immiscible solvent and precipitation of the oxidase with a salt. According to the reference, the preferred solvent used is n-butanol and suitable salts for precipitation of cholestrol oxidase include ammonium sulfate, magnesium sulfate, sodium sulfate, potassium sulfate and the like.
Although the above described methods may be used to purify peroxidase enzymes, they are not ideally suited for all applications in that they are either costly, or require the addition of reactive materials which precipitates out either the peroxidase enzyme or the contaminant. Accordingly, there exists a need for a simple and cost efficient method for removing impurities from solutions containing peroxidase enzymes.